The allergic inflammatory reaction in neonatal streptozotocininduced diabetic rats: evidence of insulin resistance and microvascular dysfunction

To investigate the allergic reaction in neonatal streptozotocin (nSTZ)-induced diabetes mellitus. Male newborn Wistar rats were made diabetic by the injection of streptozotocin (160 mg/kg, i. p.) and used 8 weeks thereafter. Animals were sensitized against ovalbumin (OA, 50 mu g and Al(OH)3, 5 mg, s. c.) and challenged 14 or 21 days thereafter. OA-induced airway inflammation and OA-induced pleurisy models were used to investigate leukocyte migration (total and differential leukocyte counts) and lung vascular permeability (Evans blue dye extravasation). nSTZ-diabetic rats presented glucose intolerance and insulin resistance. Relative to controls, nSTZ rats exhibited a 30% to 50% reduction in lung vascular permeability. Leukocyte infiltration in both models of allergen-induced inflammation, and number of pleural mast cells did not differ between groups. Data suggest that the reduction of allergic inflammatory reactions in nSTZ rats is restricted to microvascular dysfunctions and associated, probably, with insulin resistance in lung microvascular endothelium.

Differential Antitumor Effects of IgG and IgM Monoclonal Antibodies and Their Synthetic Complementarity-Determining Regions Directed to New Targets of B16F10-Nex2 Melanoma Cells

Malignant melanoma has increased incidence worldwide and causes most skin cancer-related deaths. A few cell surface antigens that can be targets of antitumor immunotherapy have been characterized in melanoma. This is an expanding field because of the ineffectiveness of conventional cancer therapy for the metastatic form of melanoma. In the present work, antimelanoma monoclonal antibodies (mAbs) were raised against B16F10 cells (subclone Nex4, grown in murine serum), with novel specificities and antitumor effects in vitro and in vivo. MAb A4 (IgG2ak) recognizes a surface antigen on B16F10-Nex2 cells identified as protocadherin beta(13). It is cytotoxic in vitro and in vivo to B16F10-Nex2 cells as well as in vitro to human melanoma cell lines. MAb A4M (IgM) strongly reacted with nuclei of permeabilized murine tumor cells, recognizing histone 1. Although it is not cytotoxic in vitro, similarly with mAb A4, mAb A4M significantly reduced the number of lung nodules in mice challenged intravenously with B16F10-Nex2 cells. The V(H) CDR3 peptide from mAb A4 and V(L) CDR1 and CDR2 from mAb A4M showed significant cytotoxic activities in vitro, leading tumor cells to apoptosis. A cyclic peptide representing A4 CDR H3 competed with mAb A4 for binding to melanoma cells. MAb A4M CDRs L1 and L2 in addition to the antitumor effect also inhibited angiogenesis of human umbilical vein endothelial cells in vitro. As shown in the present work, mAbs A4 and A4M and selected CDR peptides are strong candidates to be developed as drugs for antitumor therapy for invasive melanoma.

Tumor-derived microvesicles modulate the establishment of metastatic melanoma in a phosphatidylserine-dependent manner

Exposure of phosphatidylserine (PS) on cellular membranes and membrane-derived microvesicles stimulates a number of anti-inflammatory responses involved in malignant processes. Herein we show that B16F10 cells, a highly metastatic melanoma cell line, produce large quantities of PS-containing microvesicles in vitro. Tumor microvesicles increased TGF-beta(1) production by cultured macrophages and, in vivo, enhanced the metastatic potential of B16F10 cells in C57BL/6 mice, both effects being reversed by annexin V. Most strikingly, microvesicles induced melanoma metastasis in BALB/c mice, which are normally resistant to this tumor cell line. Altogether, this is the first demonstration that tumor-derived microvesicles favor the establishment of melanoma metastasis in a PS-dependent manner, possibly by down-regulating the host`s inflammatory and/or anti-tumoral immune responses. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

The geodiamolide H, derived from Brazilian sponge Geodia corticostylifera, regulates actin cytoskeleton, migration and invasion of breast cancer cells cultured in three-dimensional environment

We are investigating effects of the depsipeptide geodiamolide H, isolated from the Brazilian sponge Geodia corticostylifera, on cancer cell lines grown in 3D environment. As shown previously geodiamolide H disrupts actin cytoskeleton in both sea urchin eggs and breast cancer cell monolayers. We used a normal mammary epithelial cell line MCF 10A that in 3D assay results formation of polarized spheroids. We also used cell lines derived from breast tumors with different degrees of differentiation: MCF7 positive for estrogen receptor and the Hs578T, negative for hormone receptors. Cells were placed on top of Matrigel. Spheroids obtained from these cultures were treated with geodiamolide H. Control and treated samples were analyzed by light and confocal microscopy. Geodiamolide H dramatically affected the poorly differentiated and aggressive Hs578T cell line. The peptide reverted HsS78T malignant phenotype to polarized spheroid-like structures. MCF7 cells treated by geodiamolide H exhibited polarization compared to controls. Geodiamolide H induced striking phenotypic modifications in Hs578T cell line and disruption of actin cytoskeleton. We investigated effects of geodiamolide H on migration and invasion of Hs578T cells. Time-lapse microscopy showed that the peptide inhibited migration of these cells in a dose-dependent manner. Furthermore invasion assays revealed that geodiamolide H induced a 30% decrease on invasive behavior of Hs578T cells. Our results suggest that geodiamolide H inhibits migration and invasion of Hs578T cells probably through modifications in actin cytoskeleton. The fact that normal cell lines were not affected by treatment with geodiamolide H stimulates new studies towards therapeutic use for this peptide.

Adhesion and protease activity in cell lines from human salivary gland tumors are regulated by the laminin-derived peptide AG73, syndecan-1 and beta 1 integrin

We studied the induction of protease activity by the laminin alpha 1-derived peptide AG73 in cells from adenoid cystic carcinoma (CAC2) and myoepithelioma (M1), respectively a malignant and a benign salivary gland tumors. Laminin alpha 1 chain and MMP9 were immunolocalized in adenoid cystic carcinoma and myoepithelioma in vivo and in vitro. Cells grown inside AG73-enriched laminin-111 exhibited large spaces in the extracellular matrix, suggestive of remodeling. The broad spectrum MMP inhibitor GM6001 decreased spaces induced by AG73 in CAC2 and M I cells. This result strongly suggests that AG73-mediated matrix remodeling involves matrix metalloproteinases. CAC2 and M1 cells cultured on AG73 showed a dose-dependent increase of MMP9 secretion, as detected by zymography. Furthermore, siRNA silencing of MMP9 decreased remodeling in 3D cultures. We searched for AG73 receptors regulating MMP9 activity in our cell lines. CAC2 and M1 cells grown on AG73 exhibited colocalization of syndecan-1 and beta 1 integrin. siRNA knockdown of syndecan-1 expression in these cells resulted in decreased adhesion to AG73 and reduced protease and remodeling activity. We investigated syndecan-1 co-receptors in both cell lines. Silencing beta 1 integrin inhibited adhesion to AG73, matrix remodeling and protease activity. Double-knockdown experiments were carried out to further explore syndecan-1 and beta 1 integrin cooperation. CAC2 cells transfected with both syndecan-1 and beta 1 integrin siRNA oligos showed significant decrease in adhesion to AG73. Simultaneous silencing of receptors also induced a decrease in protease activity. Our results suggest that syndecan-1 and beta 1 integrin signaling downstream of AG73 regulate adhesion and MMP production by CAC2 and M1 cells. (c) 2008 Elsevier B.V./International Society of Matrix Biology. All rights reserved.

The novel ruthenium-gamma-linolenic complex [Ru(2)(aGLA)(4)Cl] inhibits C6 rat glioma cell proliferation and induces changes in mitochondrial membrane potential, increased reactive oxygen species generation and apoptosis in vitro

The present study reports the synthesis of a novel compound with the formula [Ru(2)(aGLA)(4)Cl] according to elemental analyses data, referred to as Ru(2)GLA. The electronic spectra of Ru(2)GLA is typical of a mixed valent diruthenium(II,III) carboxylate. Ru(2)GLA was synthesized with the aim of combining and possibly improving the anti-tumour properties of the two active components ruthenium and gamma-linolenic acid (GLA). The properties of Ru(2)GLA were tested in C6 rat glioma cells by analysing cell number, viability, lipid droplet formation, apoptosis, cell cycle distribution, mitochondrial membrane potential and reactive oxygen species. Ru(2)GLA inhibited cell proliferation in a time and concentration dependent manner. Nile Red staining suggested that Ru(2)GLA enters the cells and ICP-AES elemental analysis found all increase in ruthenium from <0.02 to 425 mg/Kg in treated cells. The sub-G1 apoptotic cell population was increased by Ru(2)GLA (22 +/- 5.2%) when analysed by FACS and this was confirmed by Hoechst staining of nuclei. Mitochondrial membrane potential was decreased in the presence of Ru(2)GLA (44 +/- 2.3%). In contrast, the cells which maintained a high mitochondrial membrane potential had an increase (18 +/- 1.5%) in reactive oxygen species generation. Both decreased mitochondrial membrane potential and increased reactive oxygen species generation may be involved in triggering apoptosis in Ru(2)GLA exposed cells. The EC(50) for Ru(2)GLA decreased with increasing time of exposure from 285 mu M at 24h, 211 mu M at 48 h to 81 mu M at 72 h. In conclusion, Ru(2)GLA is a novel drug with anti proliferative properties in C6 glioma cells and is a potential candidate for novel therapies in gliomas. Copyright (C) 2009 John Wiley & Sons, Ltd.

Noninvasive Evaluation of Oral Lesions Using Depth-sensitive Optical Spectroscopy

BACKGROUND: Optical spectroscopy is a noninvasive technique with potential applications for diagnosis of oral dysplasia and early cancer. In this study, we evaluated the diagnostic performance of a depth-sensitive optical spectroscopy (DSOS) system for distinguishing dysplasia and carcinoma from non-neoplastic oral mucosa. METHODS: Patients with oral lesions and volunteers without any oral abnormalities were recruited to participate. Autofluorescence and diffuse reflectance spectra of selected oral sites were measured using the DSOS system. A total of 424 oral sites in 124 subjects were measured and analyzed, including 154 sites in 60 patients with oral lesions and 270 sites in 64 normal volunteers. Measured optical spectra were used to develop computer-based algorithms to identify the presence of dysplasia or cancer. Sensitivity and specificity were calculated using a gold standard of histopathology for patient sites and clinical impression for normal volunteer sites. RESULTS: Differences in oral spectra were observed in: (1) neoplastic versus nonneoplastic sites, (2) keratinized versus nonkeratinized tissue, and (3) shallow versus deep depths within oral tissue. Algorithms based on spectra from 310 nonkeratinized anatomic sites (buccal, tongue, floor of mouth, and lip) yielded an area under the receiver operating characteristic curve of 0.96 in the training set and 0.93 in the validation set. CONCLUSIONS: The ability to selectively target epithelial and shallow stromal depth regions appeared to be diagnostically useful. For nonkeratinized oral sites, the sensitivity and specificity of this objective diagnostic technique were comparable to that of clinical diagnosis by expert observers. Thus, DSOS has potential to augment oral cancer screening efforts in community settings. Cancer 2009;115:1669-79. (C) 2009 American Cancer Society.

Non-homogeneous liver distribution of photosensitizer and its consequence for photodynamic therapy outcome

Background: Photodynamic therapy is mainly used for treatment of malignant lesions, and is based on selective location of a photosensitizer in the tumor tissue, followed by light at wavelengths matching the photosensitizer absorption spectrum. In molecular oxygen presence, reactive oxygen species are generated, inducing cells to die. One of the limitations of photodynamic therapy is the variability of photosensitizer concentration observed in systemically photosensitized tissues, mainly due to differences of the tissue architecture, cell lines, and pharmacokinetics. This study aim was to demonstrate the spatial distribution of a hematoporphyrin derivative, Photogem(R), in the healthy liver tissue of Wistar rats via fluorescence spectroscopy, and to understand its implications on photodynamic response. Methods: Fifteen male Wistar rats were intravenously photosensitized with 1.5 mg/kg body weight of Photogem(R). Laser-induced fluorescence spectroscopy at 532nm-excitation was performed on ex vivo liver slices. The influence of photosensitizer surface distribution detected by fluorescence and the induced depth of necrosis were investigated in five animals. Results: Photosensitizer distribution on rat liver showed to be greatly non-homogeneous. This may affect photodynamic therapy response as shown in the results of depth of necrosis. Conclusions: As a consequence of these results, this study suggests that photosensitizer surface spatial distribution should be taken into account in photodynamic therapy dosimetry, as this will help to better predict clinical results. (C) 2010 Elsevier B.V. All rights reserved.

Intrinsically Multivariate Predictive Genes

Canalizing genes possess such broad regulatory power, and their action sweeps across a such a wide swath of processes that the full set of affected genes are not highly correlated under normal conditions. When not active, the controlling gene will not be predictable to any significant degree by its subject genes, either alone or in groups, since their behavior will be highly varied relative to the inactive controlling gene. When the controlling gene is active, its behavior is not well predicted by any one of its targets, but can be very well predicted by groups of genes under its control. To investigate this question, we introduce in this paper the concept of intrinsically multivariate predictive (IMP) genes, and present a mathematical study of IMP in the context of binary genes with respect to the coefficient of determination (CoD), which measures the predictive power of a set of genes with respect to a target gene. A set of predictor genes is said to be IMP for a target gene if all properly contained subsets of the predictor set are bad predictors of the target but the full predictor set predicts the target with great accuracy. We show that logic of prediction, predictive power, covariance between predictors, and the entropy of the joint probability distribution of the predictors jointly affect the appearance of IMP genes. In particular, we show that high-predictive power, small covariance among predictors, a large entropy of the joint probability distribution of predictors, and certain logics, such as XOR in the 2-predictor case, are factors that favor the appearance of IMP. The IMP concept is applied to characterize the behavior of the gene DUSP1, which exhibits control over a central, process-integrating signaling pathway, thereby providing preliminary evidence that IMP can be used as a criterion for discovery of canalizing genes.

Identification of protein-coding and intronic noncoding RNAs down-regulated in clear cell renal carcinoma

The clear cell subtype of renal cell carcinoma (RCC) is the most lethal and prevalent cancer of the urinary system. To investigate the molecular changes associated with malignant transformation in clear cell RCC, the gene expression profiles of matched samples of tumor and adjacent non-neoplastic tissue were obtained from six patients. A custom-built cDNA microarray platform was used, comprising 2292 probes that map to exons of genes and 822 probes for noncoding RNAs mapping to intronic regions. Intronic transcription was detected in all normal and neoplastic renal tissues. A subset of 55 transcripts was significantly down-regulated in clear cell RCC relative to the matched nontumor tissue as determined by a combination of two statistical tests and leave-one-out patient cross-validation. Among the down-regulated transcripts, 49 mapped to untranslated or coding exons and 6 were intronic relative to known exons of protein-coding genes. Lower levels of expression of SIN3B, TRIP3, SYNJ2BP and NDE1 (P<0.02), and of intronic transcripts derived from SND1 and ACTN4 loci (P<0.05), were confirmed in clear cell RCC by Real-time RT-PCR. A subset of 25 transcripts was deregulated in additional six nonclear cell RCC samples, pointing to common transcriptional alterations in RCC irrespective of the histological subtype or differentiation state of the tumor. Our results indicate a novel set of tumor suppressor gene candidates, including noncoding intronic RNAs, which may play a significant role in malignant transformations of normal renal cells. (C) 2008 Wiley-Liss, Inc.

Expression of human papillomavirus type 16 E7 oncoprotein alters keratinocytes expression profile in response to tumor necrosis factor-alpha

Acute expression of E7 oncogene from human papillomavirus (HPV) 16 or HPV18 is sufficient to overcome tumor necrosis factor (TNF)-alpha cytostatic effect on primary human keratinocytes. In the present study, we investigated the molecular basis of E7-induced TNF resistance through a comparative analysis of the effect of this cytokine on the proliferation and global gene expression of normal and E7-expressing keratinocytes. Using E7 functional mutants, we show that E7-induced TNF resistance correlates with its ability to mediate pRb degradation and cell transformation. On the other hand, this effect does not depend on E7 sequences required to override DNA damage-induced cell cycle arrest or extend keratinocyte life span. Furthermore, we identified a group of 66 genes whose expression pattern differs between normal and E7-expressing cells upon cytokine treatment. These genes are mainly involved in cell cycle regulation suggesting that their altered expression may contribute to sustained cell proliferation even in the presence of a cytostatic stimulus. Differential expression of TCN1 (transcobalamin I), IFI44 (Interferon-induced protein 44), HMGB2 (high-mobility group box 2) and FUS [Fusion (involved in t(12; 16) in malignant liposarcoma)] among other genes were further confirmed by western-blot and/or real-time polymerase chain reaction. Moreover, FUS upregulation was detected in HPV-positive cervical high-grade squamous intraepithelial lesions when compared with normal cervical tissue. Further evaluation of the role of such genes in TNF resistance and HPVassociated disease development is warranted.